1. Field of the Invention
The invention concerns a method for detecting an analyte in a sample using analyte-specific conjugates which have at least one heterologous group for an analyte-independent binding to a control zone. The present invention additionally provides new conjugates and reagent kits.
2. Description of the Related Art
Analyte-specific reagents such as specific binding partners for the analyte to be determined or/and analyte analogues are used to detect analytes, e.g. substances of diagnostic relevance, in a sample. In heterogeneous detection methods a solid phase is used to bind the analyte such that the analyte can be separated from other components in order to carry out a qualitative or quantitative test. This solid phase binding can be mediated by an additional analyte-specific receptor which is bound to the solid phase before or during the test procedure. In a detection method based on the principle of a sandwich test a specific binding partner for the analyte can be bound to the solid phase and the analyte is detected by means of a free binding partner that is specific for the analyte and carries a labelling group. In a competitive detection method a specific binding partner for the analyte can also be used as the solid phase analyte-specific reagent and the analyte is detected indirectly by binding a free labelled analyte analogue to the solid phase.
Test strips are a special technical embodiment of heterogeneous detection methods which contain a defined analyte detection zone for the quantitative or qualitative detection of the analyte. The free analyte-specific receptor used for the detection preferably carries a direct label i.e. a group which directly generates a signal which enables a visual qualitative evaluation of the test as well as an instrument-based quantitative determination.
In addition to the analyte detection zone, diagnostic test strips also contain a control zone which allows the user to differentiate between a negative test result and an incorrect use or functional defect of the test. This control zone is usually designed such that it enables the free analyte-specific receptor to be immobilized independently of the presence of the analyte in the sample. Hence this control zone in any case becomes coloured when the test functions correctly.
The binding of the free analyte-specific receptor in the control zone can be achieved by various methods. If the free analyte-specific receptor is a specific binding partner for the analyte, then the analyte, an analyte analogue or an epitope of the analyte can be immobilized in the control zone which captures the free analyte binding partner. If the free binding partner is an antibody, an antibody, antigen analogue or an epitope of the antigen that reacts with the antibody can therefore be used for the control zone.
However, this embodiment of the control zone cannot be realised in some cases because the analyte is not available in adequate amounts or/and is not sufficiently characterized in order to prepare analogues or/and epitopes thereof or/and the specific binding partner recognizes regions of the analyte that cannot be transformed into an epitope or which are no longer accessible after immobilization on the test strip.
In such cases a receptor directed against a homologous non-analyte binding region of the binding partner has been previously used in order to capture the free analyte-specific binding partner in the control zone of the test strip. If the binding partner is an antibody, anti-antibodies which recognize the constant region of the detection antibody or other reagents that can specifically bind to immunoglobulins such as protein A or protein G have been used as control receptors.
A signal generating reaction in the control zone is important in order to make a valid interpretation of the result of a heterogeneous detection method in a test strip format since it is only possible in this manner to check the correct function of the test strip. A coloration of only the control region should be interpreted as a negative result of a functional test-whereas an additional coloration of the analyte detection zone represents a positive test result which usually means detection of the presence of an analyte in the tested sample.
However, the binding event which ensures the coloration of the control zone may be impaired for various reasons. For example the capacity of the free analyte binding partner may be substantially exhausted by high analyte concentrations in the sample and thus it cannot or can no longer adequately bind to an analyte or epitope or analogue thereof immobilized in the control zone. The control zone then only colours weakly or not at all (Hook effect).
If other receptors that are independent of the analyte recognition of the binding partner such as antibody-binding substances, especially anti-antibodies, are used as capture reagents in the control region of the test strip, this reduces the risk of a loss of function by the Hook effect. However, this concept of test strip control has other serious disadvantages. Thus in many cases biological materials such as blood, plasma or serum are used as samples. A person skilled in the art knows that these materials contain substances that can bind antibodies which can cause interferences in an immunological test that uses antibodies as specific capture and detection reagents resulting in false-positive or false-negative results. In order to prevent such erroneous measurements, unspecific immunoglobulins of that animal species from which the specific detection antibodies were derived are usually added as components to eliminate interference. The amount of these interference-eliminating antibodies exceeds the amount of analyte-specific antibodies by several fold. They are capable of binding to the capture reagents of the control region of the test strip which are directed against the detection antibody and thus compete with the detection reagent for binding sites. Consequently smaller-amounts of the detection reagent are immobilized, the coloration of the control zone is much weaker or it does not occur at all.